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Abstract:

The present invention provides a novel cancer marker for evaluating the
onset, the preclinical stage, the clinical stage, or the prognosis of a
cancer in a subject, and an evaluation method using the same. A cancer
marker containing at least one miRNA selected from hsa-miR-92 and
hsa-miR-494 is used as a marker for cancers excluding breast cancer. A
method for evaluating the possibility of cancers excluding breast cancer
includes the step of detecting the expression level of a cancer marker in
a biological sample collected from a subject. In this method, the cancer
marker contains at least one miRNA selected from hsa-miR-92 and
hsa-miR-494.

Claims:

1. A cancer marker applicable to a cancer other than breast cancer,
comprising at least one miRNA selected from hsa-miR-92 and hsa-miR-494.

2. The cancer marker according to claim 1, wherein the hsa-miR-92 is
hsa-miR-92a.

3. The cancer marker according to claim 1, wherein the miRNA is at least
one of hsa-miR-92a and hsa-miR-494.

4. The cancer marker according to claim 1, wherein the miRNA is miRNA
contained in plasma or serum.

6. The cancer marker according to claim 1, used for judging an onset, a
preclinical stage, a clinical stage, or prognosis of the cancer.

7. An evaluation method for evaluating a possibility of a cancer other
than breast cancer, the evaluation method comprising the step of:
detecting an expression level of a cancer marker in a biological sample,
wherein the cancer marker is the cancer marker according to claim 1.

8. The evaluation method according to claim 7, wherein the hsa-miR-92 is
hsa-miR-92a.

9. The evaluation method according to claim 7, wherein the miRNA is at
least one of hsa-miR-92a and hsa-miR-494.

10. The evaluation method according to claim 7, wherein the miRNA is
miRNA contained in plasma or serum.

11. The evaluation method according to claim 7, wherein the biological
sample is a sample containing plasma or serum.

13. The evaluation method according to claim 7, further comprising the
step of: determining the possibility of the cancer based on the
expression level of the cancer marker in the biological sample detected
in the cancer marker-detecting step by at least one method selected from
the group consisting of methods (1), (2), and (3): (1) the expression
level of the cancer marker in the biological sample of a subject is
compared with an expression level of the cancer marker in a biological
sample of a normal subject, and when the expression level in the subject
is lower than the expression level in the normal subject, it is
determined that the subject has a high possibility of the cancer; (2) the
expression level of the cancer marker in the biological sample of a
subject is compared with an expression level of the cancer marker in a
biological sample of a normal subject, and as the expression level in the
subject becomes relatively lower than the expression level in the normal
subject, it is determined that the cancer in the subject is relatively
advanced; and (3) the expression level of the cancer marker in the
biological sample of a subject is compared with an expression level of
the cancer marker in a biological sample of each of cancer patients at
different progression stages, and it is determined that the cancer in the
subject is in a same progression stage as the cancer in the patient
exhibiting a same or similar expression level.

14. The evaluation method according to claim 7, wherein the expression
level of the cancer marker is represented as an amount of the cancer
marker expressed in the biological sample.

15. The evaluation method according to claim 7, further comprising the
step of: detecting an expression level of a correction marker in the
biological sample, wherein the expression level of the cancer marker is
represented as a ratio between an amount of the cancer marker expressed
in the biological sample and an amount of the correction marker expressed
in the biological sample.

16. The evaluation method according to claim 15, wherein the correction
marker is hsa-miR-638.

17. An evaluation reagent for evaluating a possibility of a cancer other
than breast cancer, the evaluation reagent comprising: a miRNA detection
reagent for detecting at least one miRNA selected from hsa-miR-92 and
hsa-miR-494, wherein the evaluation reagent is used in the evaluation
method according to claim 7.

18. The evaluation reagent according to claim 17, wherein the hsa-miR-92
is hsa-miR-92a.

19. The evaluation reagent according to claim 17, wherein the miRNA is at
least one of hsa-miR-92a and hsa-miR-494.

20. The evaluation reagent according to claim 17, further comprising: a
correction marker detection reagent for detecting a correction marker.

21. The evaluation reagent according to claim 20, wherein the correction
marker is hsa-miR-638.

22. A correction marker comprising: hsa-miR-638, wherein the correction
marker is used to correct an expression level of the cancer marker
according to claim 1 in a biological sample in the evaluation method
according to claim 15.

23. The correction marker according to claim 22, wherein the miRNA is
miRNA contained in plasma or serum.

24. A correction method for correcting an expression level of a cancer
marker in a biological sample, the correction method comprising, in order
to correct an expression level of the cancer marker according to claim 1
in a biological sample in the evaluation method according to claim 7, the
steps of: measuring an expression level of the cancer marker in a
biological sample; measuring an amount of a correction marker expressed
in the biological sample; and correcting the expression level of the
cancer marker by setting a ratio between an amount of the cancer marker
expressed and the amount of the correction marker expressed to a
corrected expression level of the cancer marker in the biological sample.

25. The correction method according to claim 24, wherein the correction
marker is the correction marker according to claim 22.

26. The correction method according to claim 24, wherein the biological
sample is a sample containing plasma or serum.

27. A correction reagent comprising: a miRNA detection reagent for
detecting hsa-miR-638, wherein the correction reagent is used to correct
an expression level of the cancer marker according to claim 1 in a
biological sample in the evaluation method according to claim 15.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a cancer marker, a method for
evaluating the possibility of cancers using the same, and an evaluation
reagent. The present invention also relates to a correction marker,
correction method, and correction reagent for correcting the cancer
marker in the evaluation method.

BACKGROUND ART

[0002] In the field of clinical medical practice, it is required to easily
judge the presence or absence of a disease, the degree of progression of
the disease, the effect obtained after a treatment, etc. Under these
circumstances, as a method for judging them indirectly, detecting a
marker whose expression amount changes specifically accompanying the
onset or progression of each disease has been proposed, and attempts
actually are made to put this into practical use.

[0003] Among various diseases, detecting malignant tumors, which are
so-called cancers, early and selecting and changing a treatment strategy
therefor appropriately are particularly important. Thus, in order to
realize indirect judgment by the detection of a marker as described
above, various cancer markers (tumor markers) have been reported.
Specific examples of the cancer marker include PSA (Prostate-Specific
Antigen), CEA (Carcinoembryonic Antigen), CA 19-9 (Carcinoembryonic
Antigen 19-9), and CA 72-4 (Carcinoembryonic Antigen 72-4). Furthermore,
it is described in Non-Patent Documents 1 and 2 that the expression of
miRNAs such as has-mir-15, has-mir-16, miR-143, and miR-145 is
downregulated in lymphocytic leukemia, colon cancer, and the like
(Non-Patent Documents 1, 2).

[0006] However, in the field of clinical medical practice, since cancer
markers with which the onset of cancers and their progression can be
judged with excellent reliability are necessary, there still is a demand
for the provision of a novel cancer marker. Thus, with the foregoing
mind, it is an object of the present invention to provide a novel cancer
marker for evaluating cancers, an evaluation method using the cancer
marker, and an evaluation reagent to be used in the evaluation method.
Furthermore, it is another object of the present invention to provide a
novel correction marker for correcting the expression level of the cancer
marker, a correction method for correcting the expression level using the
correction marker, and a correction reagent to be used in the correction
method.

Means for Solving Problem

[0007] A cancer marker of the present invention is a cancer marker
applicable to a cancer other than breast cancer, containing at least one
miRNA selected from hsa-miR-92 and hsa-miR-494.

[0008] An evaluation method of the present invention is an evaluation
method for evaluating a possibility of a cancer other than breast cancer.
The evaluation method includes the step of detecting an expression level
of a cancer marker in a biological sample. In the evaluation method, the
cancer marker is the cancer marker of the present invention.

[0009] An evaluation reagent of the present invention is an evaluation
reagent for evaluating a possibility of a cancer other than breast
cancer. The evaluation reagent contains a miRNA detection reagent for
detecting at least one miRNA selected from hsa-miR-92 and hsa-miR-494.
The evaluation reagent is used in the evaluation method of the present
invention.

[0010] A correction marker of the present invention contains hsa-miR-638.
The correction marker is used to correct an expression level of the
cancer marker of the present invention in a biological sample in the
evaluation method of the present invention.

[0011] A correction method of the present invention includes, in order to
correct an expression level of the cancer marker of the present invention
in a biological sample in the evaluation method of the present invention,
the steps of:

[0012] measuring an expression level of the cancer marker in a biological
sample;

[0013] measuring an amount of a correction marker expressed in the
biological sample; and

[0014] correcting the expression level of the cancer marker by setting a
ratio between an amount of the cancer marker expressed and the amount of
the correction marker expressed to a corrected expression level of the
cancer marker in the biological sample.

[0015] A correction reagent of the present invention contains a miRNA
detection reagent for detecting hsa-miR-638. The correction reagent is
used to correct an expression level of the cancer marker of the present
invention in a biological sample in the evaluation method of the present
invention.

Effects of the Invention

[0016] The inventors of the present invention conducted a diligent study,
and as a result, they found that the expression levels of hsa-miR-92 and
hsa-miR-494 in a biological sample decrease accompanying the development
of cancers, thereby achieving the present invention. According to the
cancer marker of the present invention, by detecting the expression level
thereof in a biological sample, it is possible to judge the presence or
absence of cancer development or the cancer progression, for example.
Furthermore, the cancer marker of the present invention provides a
significant difference between negative and positive regarding the
canceration, for example. Thus, according to the cancer marker of the
present invention, it becomes possible to detect cancers at an initial
stage easily whereas such detection is difficult by general palpation and
the like. Still further, according to the correction marker of the
present invention, the accuracy of detection using the cancer marker
further can be improved.

BRIEF DESCRIPTION OF DRAWINGS

[0017] FIGS. 1A and 1B are graphs showing the expression profiles of
various miRNAs in samples derived from normal subjects in Example 2 of
the present invention. FIG. 1A is a graph showing the signal intensity
values of various miRNAs contained in each of the samples, and FIG. 1B is
a graph showing a logarithmic value (log10[individual
rank_Normal/Average rank_Normal]) of a value obtained by dividing a
signal value rank of each miRNA in each of the samples derived from the
seven normal subjects by the signal value average rank thereof.

[0018] FIG. 2 is a graph showing the ratio (Average rank_AML/Average
rank_Normal) between the signal value average rank of each miRNA between
the samples derived from the AML patients and the signal value average
rank of each miRNA among the samples derived from the normal subjects in
Example 2 of the present invention.

[0019] FIG. 3 is a graph showing the ratio (hsa-miR-92a/hsa-miR-638)
between the fluorescence intensity of hsa-miR-92a as a cancer marker and
the fluorescence intensity of hsa-miR-638 a correction marker in samples
derived from normal subjects and in samples derived from acute leukemia
patients in Example 3 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Cancer Marker

[0020] The cancer marker of the present invention is, as described above,
a cancer marker applicable to a cancer other than breast cancer,
containing at least one miRNA selected from hsa-miR-92 and hsa-miR-494.

[0021] As described above, by the inventors of the present invention, it
has been revealed that the expression levels of these miRNAs change
specifically accompanying the canceration of cells of various tissues.
More specifically, it has been revealed that the expression levels of the
miRNAs in, for example, plasma or serum decreases accompanying the
canceration. From this fact, it is interpreted that, for example, the
expression levels of the miRNAs decrease significantly: after the onset
of a cancer as compared with before the onset of the cancer; in the
preclinical stage as compared with before the preclinical stage; in the
clinical stage as compared with before the clinical stage, in the initial
stage as compared with before the initial stage; after the initial stage
as compared with in the initial stage. Therefore, by detecting the
expression levels of these miRNAs, it becomes possible to carry out
evaluation or the like of the possibility that a subject may develop a
cancer, whether or not canceration has occurred, the stage of cancer
progression such as a preclinical stage (an initial stage) or a clinical
stage (an advanced stage or a disease stage), or prognosis, for example.

[0022] The meaning of the terms used in the present invention is as
follows. The term "cancer" generally means malignant tumor. The term
"canceration" generally means the onset of a cancer and also encompasses
"malignant transformation". Regarding the term "onset", for example, the
time point at which one is diagnosed as having a specific disease through
synthetic judgment based on disease-specific clinical symptoms, test
data, etc., is referred to as the onset of the disease. The term
"preclinical stage" generally refers to a condition before the onset of a
disease where disease-specific clinical symptoms have not appeared yet
but in an early stage of the disease in which a trace amount of malignant
tumor cells are present already. In cervical cancer, the preclinical
stage generally indicates precancerous lesions such as CIN1, CIN2, and
CIN3. In colon cancer, the preclinical stage indicates a condition of
having adenoma. The term "clinical stage" generally refers to cancers
found through image findings and cancers that can be identified by the
use of existing tumor markers. The term "initial stage" generally
indicates a condition of having an early cancer. The term "prognosis"
means, for example, a postoperative course of a disease. Since the cancer
marker of the present invention can provide useful information for, for
example, predicting prognosis, foreseeing the course of a disease, and
selecting an appropriate treatment method, it also can be referred to as
a "prognostic factor". The "stage of cancer progression" can be judged as
appropriate based on, for example, the kind of cancerous tissues or the
like. In general, Stage 0 and Stage I can be classified as an initial
cancer, Stage II can be classified as an early cancer, and Stage III and
Stage IV can be classified as an advanced cancer.

[0024] In the present invention, each miRNA may be, for example, a single
strand (monomer) or a double strand (dimer). Furthermore, in the present
invention, each miRNA preferably is the miRNA in its mature form cleaved
by ribonuclease such as Dicer.

[0025] In the present invention, the hsa-miR-92 may be, for example,
hsa-miR-92a, which preferably is the miRNA in its mature form as
described above.

[0026] The sequence of the mature hsa-miR-92a is registered under
Accession No. MIMAT0000092, for example. This sequence is shown as SEQ ID
NO: 1 below.

[0027] The hsa-miR-494 may be, for example, the miRNA in its mature form
as described above. The sequence of the mature miRNA is registered under
Accession No. MIMAT0002816. The sequence of the mature hsa-miR-494 is
shown as SEQ ID NO: 2.

[0035] The miRNAs in the present invention encompass, for example,
polynucleotides having a base sequence with a homology to the base
sequences of the respective SEQ ID NOs: 1 and 2, and polynucleotides
having a base sequence complementary thereto. The "homology" refers to
the degree of identity between sequences to be compared with each other
when they are aligned appropriately, and represents the occurrence ratio
(%) of perfect match of amino acids between these sequences. When it is
described that the base sequence of a polynucleotide "has a homology" to
the base sequences of the miRNAs of the present invention, it means that
the polynucleotide is similar enough to the miRNAs to be able to maintain
the function as the miRNAs of the present invention. The alignment can be
achieved by using an arbitrary algorithm such as BLAST, for example. Even
when the base sequences differ from each other by, for example, point
mutation, deletion, or addition, it can be said that they are homologous
as long as such a difference does not affect the function of the miRNAs.
The number of bases different between the base sequences is, for example,
1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. Furthermore,
when base sequences of two polynucleotides to be compared with each other
have an identity of, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or about 99%, it can be said that they are homologous.
Furthermore, for example, when one of the two polynucleotides hybridizes
to a polynucleotide having a base sequence complementary to the other
polynucleotide under stringent conditions, it can be said that the two
polynucleotides are homologous. The stringent conditions are not
particularly limited, and may be such that, for example, the two
polynucleotides are kept at a temperature of "Tm-25° C." overnight
in a solution containing 6×SSC, 0.5% SDS, 5×Denhardt's
solution, and 0.01% denatured salmon sperm nucleic acid.

[0036] The cancer marker of the present invention is, for example, a miRNA
present in a biological sample. The biological sample in the present
invention is a liquid fraction collected from a living organism (e.g., a
subject), and examples thereof include blood, saliva, urine, and diluted
solutions thereof. Among them, a blood sample is preferable because
change in expression levels of the various miRNAs accompanying the
canceration is particularly noticeable. The blood sample is not
particularly limited. It may be, for example, a sample containing a
liquid fraction of blood, such as a so-called plasma-containing sample or
serum-containing sample. Specific examples of the sample include whole
blood, a liquid fraction, plasma fraction, and serum fraction of whole
blood, samples containing them, and samples obtained by diluting them.

[0037] <Evaluation Method>

[0038] The evaluation method of the present invention is, as described
above, an evaluation method for evaluating a possibility of a cancer
other than breast cancer. The evaluation method includes the step of
detecting an expression level of a cancer marker in a biological sample.
In the evaluation method, the cancer marker is the cancer marker of the
present invention.

[0039] The present invention is characterized in that the cancer marker of
the present invention is detected as a cancer marker, and for example, a
method for detecting the expression level of each miRNA is by no means
limited. The cancer marker of the present invention is as described
above. In the present invention, the cancer marker to be detected may be,
for example, either one of hsa-miR-92 and hsa-miR-494 or both of them.
When the cancer marker to be detected is hsa-miR-92, it is preferable
that the hsa-miR-92 is in its mature form, for example. When the cancer
marker to be detected is hsa-miR-92a, it is preferable that the
hsa-miR-92a is in its mature form.

[0041] In the present invention, the biological sample is not particularly
limited, and examples thereof include, as described above, blood, saliva,
and urine. Among them, the biological sample preferably is the
above-described blood sample, more preferably a sample containing plasma,
a sample containing serum, or the like, because change in expression
levels of the various miRNAs accompanying the canceration is particularly
noticeable. Such a blood sample is particularly preferable also because,
for example, a test can be carried out merely by collecting blood and the
pain and burden placed on a subject can be reduced considerably.

[0042] The evaluation method of the present invention further may include
the step of determining the possibility of the cancer. More specifically,
the evaluation method of the present invention may include the step of
determining the possibility of the cancer based on the expression level
of the cancer marker in the biological sample detected in the cancer
marker-detecting step by at least one method selected from the group
consisting of methods (1), (2), and (3):

(1) the expression level of the cancer marker in the biological sample of
a subject is compared with an expression level of the cancer marker in a
biological sample of a normal subject, and when the expression level in
the subject is lower than the expression level in the normal subject, it
is determined that the subject has a high possibility of the cancer; (2)
the expression level of the cancer marker in the biological sample of a
subject is compared with an expression level of the cancer marker in a
biological sample of a normal subject, and as the expression level in the
subject becomes relatively lower than the expression level in the normal
subject, it is determined that the cancer in the subject is relatively
advanced; and (3) the expression level of the cancer marker in the
biological sample of a subject is compared with an expression level of
the cancer marker in a biological sample of each of cancer patients at
different progression stages, and it is determined that the cancer in the
subject is in the same progression stage as the cancer in the patient
exhibiting the same or similar expression level.

[0043] In the present invention, the possibility of a cancer encompasses,
for example, the possibility that the subject may develop a cancer,
whether or not canceration has occurred, the stage of cancer progression
such as a preclinical stage or a clinical stage, prognosis, or the like.
In the present invention, the term "normal subject" means, for example, a
subject who is not judged as having developed a cancer to be evaluated or
having a possibility that he has developed the cancer.

[0044] The expression level of the cancer marker in the normal subject in
the methods (1) and (2) can be determined previously using a biological
sample collected from the normal subject, for example. Furthermore, the
expression levels of the cancer marker in the cancer patients in the
method (3) also can be determined by, for example, previously classifying
the patients according to the progression stage and using biological
samples collected from the patients at the respective progression stages.
In the methods (1) to (3), the kind of the biological samples of the
normal subject and the patients preferably are the same as the kind of
the biological sample of the subject, for example, and the biological
sample preferably is a blood sample, particularly preferably a sample
containing plasma or a sample containing serum, for example. Furthermore,
the biological samples of the normal subject and the patients preferably
are prepared in the same manner and under the same conditions as the
biological sample of the subject, for example.

[0045] In the present invention, the expression level of the cancer marker
may be, for example, the amount of the cancer marker expressed in the
biological sample. The amount of the cancer marker expressed may be, for
example, the actual amount of miRNA or a value correlated with the actual
amount of miRNA. Examples of the latter include a signal value obtained
when detecting the miRNA. This signal value can be determined as
appropriate depending on, for example, the method for detecting the miRNA
and the kind of a detector for detecting the signal value. When the
detection method is, for example, a PCR method such as a real-time RT-PCR
(real-time reverse transcription-polymerase chain reaction) method, the
signal value can be expressed with a unit of copies/μl or the like,
for example.

[0046] It is preferable that the expression level of the cancer marker is
corrected with the expression level of a correction marker, for example.
The correction marker is not particularly limited, and may be, for
example, a substance exhibiting a constitutive expression level in a
biological sample. In particular, the correction marker preferably is a
substance exhibiting a constitutive expression level in a biological
sample regardless of the presence or absence of canceration. By detecting
the expression level of such a correction marker as an internal standard
in a biological sample collected to detect the cancer marker, the
expression levels of the cancer marker between different subjects and the
expression levels in the same subject over time can be compared with
still higher reliability, for example. For the reasons stated above, the
evaluation method of the present invention further may include the step
of detecting the expression level of the correction marker in the
biological sample collected from the subject.

[0047] The above-described corrected expression level of the cancer marker
can be represented as the ratio between the amount of the cancer marker
expressed and the amount of the correction marker expressed in the
biological sample, for example. Specifically, it can be represented as,
for example, "the amount of the cancer marker expressed/the amount of the
correction marker expressed" or "the amount of the correction marker
expressed/the amount of the cancer marker expressed". The expression of
the cancer marker of the present invention decreases accompanying the
canceration. Thus, in the case where it is represented as "the amount of
the cancer marker expressed/the amount of the correction marker
expressed" as the former, for example, it can be judged that the
possibility of a cancer becomes higher as the value becomes relatively
smaller. On the other hand, in the case where it is represented as "the
amount of the correction marker expressed/the amount of the cancer marker
expressed" as the latter, it can be judged that the possibility of a
cancer becomes higher as the value becomes greater, for example.

[0048] The correction marker is not particularly limited, and a known
marker can be used as the correction marker. In the present invention, it
is preferable to use, in particular, hsa-miR-638 as the correction
marker. The correction marker containing this miRNA corresponds to the
correction marker of the present invention to be described later, and it
exhibits constitutive expression in a biological sample regardless of the
presence or absence of canceration, for example. Since the correction
marker of the present invention exhibits constitutive expression
regardless of the presence or absence of canceration as described above,
it can serve as an internal standard in a biological sample.

[0049] The correction marker of the present invention, i.e., the
hsa-miR-638, preferably is the miRNA in its mature form cleaved by a
ribonuclease such as Dicer, for example.

[0050] The hsa-miR-638 may be, for example, the miRNA in its mature form
as described above. The sequence of the mature miRNA is registered under
Accession No. MIMAT00033088. The sequence of the mature hsa-miR-638 is
shown as SEQ ID NO: 3. hsa-miR-638 (SEQ ID NO: 3)

[0051] 5'-agggaucgcgggcggguggcggccu-3'

[0052] In the present invention, the subject is not particularly limited,
and examples thereof include: mammals including humans, dogs, and cats;
primates; and rodents.

[0053] In the following, the evaluation method of the present invention
will be described with reference to an example where plasma or serum is
used as a biological sample. It is to be noted, however, the present
invention is not limited thereto.

[0054] First, total RNA is extracted from plasma or serum of a subject.
The plasma or serum can be recovered by a known method, and for example,
it can be prepared by collecting whole blood from the subject and
removing a blood cell fraction through centrifugation. Furthermore, the
method for extracting the total RNA from the plasma or serum is not
particularly limited, and any of known methods can be employed. Examples
of the extraction method include a guanidine-CsCl ultracentrifugation
method and an AGPC (Acid Guanidinium-Phenol-Chloroform) method.

[0055] Next, the cancer marker of the present invention, i.e., at least
one of hsa-miR-92 and hsa-miR-494, in the extracted total RNA is
detected. The method for detecting the cancer marker of the present
invention is by no means limited, as long as it can detect the expression
of the miRNA as described above. Specific examples of the detection
method include, for example, Northern blot analysis, a real-time RT-PCR
detection method, and a microarray analysis method. The present invention
is characterized in that the miRNA to be detected as a cancer marker is
at least one of hsa-miR-92 and hsa-miR-494. A method for detecting it is
by no means limited, and those skilled in the art can carry out such a
method based on common general technical knowledge.

[0056] Northern blot analysis can be carried out, for example, using a
probe as described below. The probe is not particularly limited, and
probes that can detect the above-described miRNAs can be used. For
example, the probe may be the one that can hybridize to any of the
above-described miRNAs. A commercially available product may be used as
the probe, or the probe may be prepared on your own, for example. The
sequence of the probe can be designed as appropriate based on the base
sequences of the miRNAs and common general technical knowledge, for
example. Specific examples of the probe include the one having a sequence
complementary to the miRNA to be detected. It is preferable that the
sequence of the probe is, for example, at least about 70% complementary
to the miRNA to be detected, more preferably at least 90% complementary
to the same, and particularly preferably 100% complementary to the same.
The constitutional unit of the probe is not particularly limited, and,
for example, a known constitutional unit can be employed. Specific
examples of the constitutional unit include nucleotides such as
deoxyribonucleotide and ribonucleotide, PNA (Peptide Nucleic Acid), and
LNA (Locked Nucleic Acid). Examples of the LNA include BNA (Bridged
Nucleic Acid) such as 2',4'-bridged nucleic acid. Bases in the
nucleotides are not particularly limited. They may be, for example,
natural bases such as adenine, guanine, cytosine, thymine, and uracil, or
may be unnatural bases (artificial bases). The length of the probe is not
particularly limited, and is, for example, 10 to 100 mer, preferably 10
to 40 mer, more preferably 10 to 25 mer, and still more preferably 15 to
20 mer.

[0057] As a specific example, first, the extracted total RNA is
fractionated depending on the strand length by electrophoresis, and the
fractionated total RNA is transcribed onto a membrane from the gel used
in the electrophoresis. Examples of the membrane include a nitrocellulose
membrane and a nylon membrane. Subsequently, the membrane on which the
fractionated RNAs have been transcribed was incubated in a predetermined
buffer in the presence of the above-described probe labeled with a
radioactive material such as 32P. Then, by detecting the label of
the probe, miRNA that has hybridized with the probe can be detected. When
the probe labeled with a radioactive material is used as described above,
the miRNA that has hybridized with the probe can be quantified by, for
example, autoradiography, based on the band intensity. Conditions of the
Northern blot analysis are not particularly limited, and it is preferable
that, for example, prehybridization, hybridization, and washing are
carried out under stringent conditions. For example, in the case where
DNA labeled with 32P is used as a probe, the stringent conditions
are at 37° C. in a hybridization buffer. The hybridization buffer
may be, for example, a buffer containing 0.25 mol/l sodium phosphate (pH
7.2), 7% SDS, and 0.5% sodium pyrophosphate. Furthermore, in the washing
step, the stringent conditions are at 37° C. in a washing buffer
containing 2×SSC and 1% SDS and further at room temperature in a
washing buffer containing 0.1×SSC. It should be note that the
stringent conditions are not limited thereto, and depending on the
selected detection method, conditions standard therefor can be employed.

[0058] The real-time RT-PCR detection method can be carried out using a
fluorescent reagent as described below, for example. First, for example,
a linker is ligated to each of 5' end and 3' end of the extracted total
RNA. Then, with the total RNA having the linker ligated thereto as a
template, cDNA is amplified. Further, with the thus-obtained cDNA as a
template, PCR is carried out using a primer that can amplify the miRNA to
be detected. The primer is by no means limited, and examples thereof
include primers that can hybridize to the above-described miRNA or a
peripheral region of the miRNA. The primer can be designed as appropriate
based on the base sequence of the above-described miRNA and common
general technical knowledge, for example. Specific examples of the primer
include a probe having a sequence complementary to the miRNA to be
detected or a peripheral region of the miRNA. It is preferable that the
sequence of the primer is, for example, at least about 70% complementary
to, for example, the miRNA to be detected or a peripheral region of the
miRNA, preferably at least 80% complementary to the same, more preferably
at least 90% complementary to the same, still more preferably at least
95% complementary to the same, and particularly preferably 100%
complementary to the same. The constitutional unit of the primer is not
particularly limited, and is, for example, the same as that of the
above-described probe. The length of the primer is not particularly
limited, and may be a general length.

[0059] At the time of carrying out the PCR, it is preferable to cause a
fluorescent reagent to be present in a PCR reaction solution, for
example. Examples of the fluorescent reagent include a fluorescent dye
that specifically binds to a double-stranded nucleic acid and a
fluorescent dye that intercalates into a double-stranded nucleic acid.
When such a fluorescent reagent forms a double-stranded nucleic acid by
nucleic acid amplification, the fluorescent dye binds to or intercalates
into the double-stranded nucleic acid. Then, by measuring the
fluorescence intensity of the fluorescent dye that has bound to or
intercalated into the double-stranded nucleic acid, it is possible to
quantify the miRNA to be detected. Examples of the fluorescent dye
include SYBR (trademark) Green. Such a real-time RT-PCR detection method
can be carried out by a known method, for example. Also, it can be
carried out, for example, using a commercially available reagent such as
SYBR (trademark) Green PCR Master Mix (trade name, Perkin-Elmer Applied
Biosystems) and a commercially available detector such as ABI Prism 7900
Sequence Detection System (trade name, Perkin-Elmer Applied Biosystems)
in accordance with their manuals. Furthermore, the above-described primer
also may serve as the fluorescent reagent. Such a primer may be, for
example, a labeled primer labeled with a fluorescent dye, whose
fluorescence is quenched when a fluorescent double-stranded nucleic acid
has not yet been formed and the quenching is released when the
double-stranded nucleic acid is formed. Such a labeled primer can be
designed based on common general technical knowledge, for example.

[0060] When the expression amount is measured by such a detection method,
it is preferable that an internal standard such as a correction marker is
measured, and with regard to the miRNA to be detected, the expression
amount corrected with the internal standard is calculated as will be
described later, for example.

[0061] On the other hand, the cancer marker of the present invention in
plasma or serum collected from a normal subject also is detected in the
same manner. At this time, it is preferable that the kind of the cancer
marker used for the subject is the same as that used for the normal
subject. It is preferable that the expression level of the cancer marker
of the present invention in the normal subject is determined previously,
for example, and it is not necessary to determine the expression level in
the normal subject every time evaluation is made. That is, it is
preferable that the previously detected expression level of the cancer
marker of the present invention in the normal subject is set to a
standard value of the normal subject. The expression level in the normal
subject may be, for example, a value obtained from a single normal
subject or may be a value calculated from the expression levels in a
plurality of normal subjects by a statistical method.

[0062] Then, from the expression level in the subject and the expression
level in the normal subject, the possibility of a cancer in the subject
can be evaluated by, for example, the following method (1) or (2).

[0063] In the method (1), for example, the expression level of the cancer
marker in the subject is compared with the expression level of the cancer
marker in the biological sample of the normal subject, and when the
expression level in the subject is significantly lower than the
expression level in the normal subject, it is determined that the
possibility of the cancer is high. As described above, the expression
level of the cancer marker of the present invention in blood decreases
accompanying the canceration, for example. Therefore, when the expression
level in the subject is equal to or higher than that in the normal
subject, it can be judged that the subject has a low possibility of
canceration. On the other hand, when the expression level in the subject
is lower than that in the normal subject, it can be judged that the
subject has a high possibility of canceration.

[0064] In the method (2), for example, the expression level of the cancer
marker in the subject is compared with the expression level of the cancer
marker in the biological sample of a normal subject, and when the
expression level in the subject becomes relatively lower than the
expression level in the normal subject, it is determined that the cancer
in the subject is relatively advanced. As described above, the expression
level of the cancer marker of the present invention in blood decreases
accompanying the progression of canceration, for example. Therefore, as
the expression level in the subject is lower than the expression level in
the normal subject and the difference in expression level between the
subject and the normal subject is relatively small, it can be judged that
the cancer progression is less severe, and as the above-described
difference is relatively large, it can be judged that the cancer
progression is severe.

[0065] In the methods (1) and (2), for example, when the expression level
in the subject is at least 50% lower than the expression level in the
normal subject, for example, it can be judged that the subject has a high
possibility of the cancer, and as the expression level in the subject
decreases further to be at least 85%, at least 90%, and at least 95%
lower than the expression level in the normal subject, it can be judged
that the cancer is advanced further. The same applies to the case where
the expression level is corrected with a correction marker as will be
described later.

[0066] Furthermore, instead of or in addition to the detection of the
cancer marker with regard to the normal subject, the cancer marker of the
present invention may be detected in the same manner with regard to serum
or plasma collected from cancer patients at different progression stages.
At this time, it is preferable that the kind of the cancer marker used
for the subject is the same as that used for the cancer patients. The
expression levels of the cancer marker of the present invention in the
patients preferably are determined previously, and it is not necessary to
determine the expression levels in the patients every time evaluation is
made. That is, it is preferable that the previously detected expression
level of the cancer marker of the present invention in each cancer
patient is set to a standard value for each progression stage. Note here
that the expression level in the cancer patient may be a value obtained
from, for example, a single cancer patient, or may be a value calculated
from the expression levels in a plurality of cancer patients by a
statistical method.

[0067] Then, from the expression level in the subject and the expression
level in each cancer patient, the possibility of the cancer in the
subject can be evaluated by, for example, the following method (3).

[0068] In the method (3), for example, the expression level of the cancer
marker in the subject is compared with the expression level of the cancer
marker in the biological sample of each of the cancer patients at
different progression stages, and it is determined that the cancer in the
subject is in the same progression stage as the cancer in the patient
exhibiting the same or similar expression level. As described above, the
expression level of the cancer marker of the present invention in blood
decreases with the progression of canceration. Therefore, by determining
the expression level in the cancer patients at different progression
stages, not only the possibility of canceration of the subject but also
the progression stage of the cancer can be evaluated through comparison
with the thus-determined expression levels.

[0069] As described in the methods (1) to (3), when comparing the
expression level in the subject with that of the normal subject or each
of the cancer patients, the significant difference therebetween can be
judged by a statistical method such as a t-test, an F-test, or a
chi-square test, for example.

[0070] Furthermore, in order to improve the reliability of the evaluation
method of the present invention, the evaluation may be carried out using
the expression level of the cancer marker of the present invention
corrected as described above.

[0071] As described above, the corrected expression level can be
represented as the ratio between the amount of the cancer marker
expressed and the amount of the correction marker expressed in a
biological sample, for example. As a specific example, it can be
represented as "the amount of the cancer marker expressed/the amount of
the correction marker expressed". In the method (1) or (2), the ratio
calculated with regard to the subject may be compared with the ratio
calculated with regard to the normal subject, for example. Furthermore,
in order to make the comparison of these ratios easier, it is preferable
that, assuming the ratio calculated with regard to the normal subject is
"1", a relative value of the ratio calculated with regard to the subject
compared to this is determined, for example. This allows the judgment to
be made more easily based on whether the calculated value with regard to
the subject is smaller or greater than "1". Furthermore, also in the
method (3), for example, the ratio calculated with regard to the subject
may be compared with the ratio calculated with regard to each of the
cancer patients at the different progression stages.

[0072] According to such an evaluation method, for example, with regard to
a subject having a cancer at a preclinical stage, it is possible to judge
that the subject has a high possibility of the cancer with high
reliability whereas such judgment has been difficult conventionally.
Furthermore, for example, the stage of cancer progression also can be
judged with high reliability. Thus, in prevention or treatments of
cancers, information important in determining strategies for medication,
operation, etc. for example, can be obtained with high reliability.

[0073] <Evaluation Reagent>

[0074] The evaluation reagent of the present invention is, as described
above, an evaluation reagent to be used in the evaluation method of the
present invention and characterized in that it contains a reagent for
detecting the cancer marker of the present invention, i.e., a miRNA
detection reagent for detecting at least one miRNA selected from
hsa-miR-92 and hsa-miR-494. According to such an evaluation reagent, it
is possible to carry out the evaluation method of the present invention
conveniently.

[0075] The present invention is characterized in that, as described above,
at least one of hsa-miR-92 and hsa-miR-494 is detected as a cancer
marker, and a method for detecting these miRNAs is by no means limited.
Therefore, it is only necessary that the miRNA detection reagent
contained in the evaluation reagent of the present invention can detect
either of these miRNAs, and for example, the kind, composition, etc. of
the reagent are by no means limited. Furthermore, those skilled in the
art can set detection reagents for these miRNAs based on common general
technical knowledge.

[0076] The evaluation reagent of the present invention further may contain
any of various enzymes, buffer solutions, washing solutions, dissolving
solutions, dispersions, diluents and the like, for example, depending on
various detection methods. Furthermore, the form of the evaluation
reagent of the present invention is not particularly limited. For
example, it may be a wet-type reagent in the liquid form or a dry-type
reagent in the dry form.

[0077] The miRNA detection reagent is not particularly limited, and
examples thereof include reagents to be used in the Northern blot
analysis and real-time RT-PCR detection method described above. Specific
examples of the miRNA detection reagent to be used in the Northern blot
analysis include labeled probes that can hybridize to either of these
miRNAs, such as those described above. Furthermore, specific examples of
the reagent to be used in the real-time RT-PCR detection method include
primers for amplifying cDNA from total RNA, primers for amplifying either
of these miRNAs, fluorescent reagents that specifically bind to or
intercalate into double-stranded nucleic acids, and various reagents that
can be used in nucleic acid amplification, such as those described above.
Examples of the various reagents include nucleotide triphosphate (dNTP)
and enzymes such as DNA polymerase.

[0078] <Evaluation Kit>

[0079] The evaluation kit of the present invention is, as described above,
an evaluation kit to be used in the evaluation method of the present
invention and characterized in that it includes a miRNA detection reagent
for detecting at least one miRNA selected from hsa-miR-92 and hsa-miR-494
(e.g., the evaluation reagent of the present invention). According to
such an evaluation kit, the evaluation method of the present invention
can be carried out conveniently. In the evaluation kit of the present
invention, the evaluation reagent of the present invention is as
described above, for example.

[0080] It is preferable that the evaluation kit of the present invention
further includes a correction marker detection reagent for detecting a
correction marker. The correction marker detection reagent is not
particularly limited, and can be determined as appropriate depending on
the kind of the correction marker, the method for detecting the
correction marker, etc. described above. As described above, the
correction marker used in the evaluation method of the present invention
preferably is the correction marker of the present invention. Thus, the
correction marker detection reagent preferably is a reagent for detecting
the correction marker of the present invention, i.e., a miRNA detection
reagent for detecting hsa-miR-638, for example. This miRNA detection
reagent is not particularly limited. As in the case of the reagent for
detecting the cancer marker of the present invention, examples of the
miRNA detection reagent include reagents to be used in the Northern blot
analysis and the real-time RT-PCR detection method.

[0081] The form of the evaluation kit of the present invention is not
particularly limited. It may be a wet-type kit in the liquid form or a
dry-type kit in the dry form. Various reagents in the evaluation kit of
the present invention may be provided separately and used together when
the kit is used, or may be mixed together before the kit is used, for
example.

[0082] The evaluation kit of the present invention may be, for example, a
test tool in which the above-described evaluation reagent of the present
invention and other various reagents are arranged. The form of the test
tool is not particularly limited, and examples of the test tool include
reactors such as a microreactor, chips such as a microchip, plates such
as a microtiter plate, and arrays such as a microarray. According to such
a test tool, for example, by detecting a necessary signal with various
kinds of existing detectors such as a real-time PCR device, the
evaluation method of the present invention can be carried out easily.
Furthermore, the test tool may include, for example, a system for
carrying out the following operations by computer processing: numerical
conversion of a detected signal; correction of a measured signal value by
the expression level of the correction marker as described above;
creation of a data file for each subject; storage of the data file in a
predetermined directory; statistical analysis of measurement results
obtained as to subjects, normal subjects, and patients; and the like.
Those skilled in the art can design such a data processing system from
existing techniques, methods, and procedures based on common general
technical knowledge.

[0083] The evaluation kit of the present invention further may include,
for example, any of various appliances and the like that can be used to
carry out the evaluation method of the present invention. Furthermore, it
is preferable that the evaluation kit of the present invention further
include instructions for use, for example.

[0084] <Correction Marker>

[0085] As described above, the correction marker of the present invention
contains hsa-miR-638 and is characterized in that it is used to correct
the expression level of the cancer marker of the present invention in a
biological sample in the evaluation method of the present invention.

[0086] The inventors of the present invention conducted a diligent study
in order to obtain a novel correction marker that serves as an internal
standard in a biological sample. As a result, they found out that the
above-described hsa-miR-638 exhibits constitutive expression in a
biological sample, in particular, in a blood sample, thus achieving the
present invention. miRNA that serves as a correction marker has not yet
been reported, and the miRNAs found by the inventors of the present
invention are the first such miRNA reported. According to such a
correction marker, for example, the comparison as to the cancer marker of
the present invention to be detected between subjects or in the same
subject over time can be carried out more reliably. The miRNA in the
correction marker of the present invention is as described above.

[0087] The correction marker of the present invention is a miRNA present
in a biological sample, and the biological sample is not particularly
limited. The biological sample is not particularly limited, and examples
thereof include, as described above, blood, saliva, and urine. Among
them, the blood samples as described above are preferable because the
expression of the correction marker therein remains constitutive
regardless of whether the subject has a cancer or not. Among the blood
samples, for example, a sample containing plasma, a sample containing
serum, a sample containing plasma and serum, and the like are more
preferable.

[0088] The correction marker of the present invention can be used to
correct the expression level of a marker to be detected in a biological
sample. Specifically, the correction marker of the present invention
preferably is used to correct the expression level of the cancer marker
of the present invention. The correction marker of the present invention
is present constitutively regardless of whether the subject has a cancer
or not, for example. Accordingly, the kind of the marker to be detected
is not limited to the cancer marker of the present invention. For
example, it may be a cancer marker whose expression level changes
accompanying the onset of a cancer. Note here that the marker to be
detected may be, for example, a marker whose expression level in a
biological sample varies depending on a disease.

[0089] <Correction Method by Correction Marker>

[0090] As described above, in order to correct the expression level of the
cancer marker of the present invention in a biological sample in the
evaluation method of the present invention, the correction method of the
present invention includes the steps of:

[0091] measuring the expression level of the cancer marker of the present
invention in a biological sample;

[0092] measuring the amount of the correction marker expressed in the
biological sample; and

[0093] correcting the expression level of the cancer marker by setting the
ratio between the amount of the cancer marker expressed and the amount of
the correction marker expressed to a corrected expression level of the
cancer marker in the biological sample.

[0094] According to the correction method of the present invention, since
the correction marker is present constitutively in a biological sample,
by correcting the expression level of the cancer marker of the present
invention in a biological sample with this correction marker, it becomes
possible to compare, for example, the expression levels of the cancer
marker between different subjects, the expression levels in the same
subject over time, etc. with still higher reliability.

[0095] It is to be noted that, as described above, the correction method
using the correction marker of the present invention can be used not only
for the cancer marker of the present invention but also for other markers
to be detected in a biological sample, for example. Since the correction
marker is present constitutively regardless of whether the subject has a
cancer or not, for example, the marker to be detected preferably is a
cancer marker whose expression level changes accompanying the onset of a
cancer. Specific examples such a cancer marker include, in addition to
the cancer marker of the present invention, for example, miR-16-1-15a,
miR-145, let-7 family, miR-155, miR-17-92 cluster, miR-21, miR-221,
miR10-b, miR-128, miR-181a, miR-181b, miR-125b, miR-145, miR-143,
miR-133b, miR-31, miR-135b, miR-96, miR-183, miR-18, miR-224, miR-199a,
miR-195, miR-200a, miR-125a, miR-122, miR-126, miR-21, miR-205, miR-15a,
miR-16-1, miR-150, miR-222, miR-103, miR-107, miR-204, miR-372, miR-373,
miR-146b, miR-197, and miR-346.

[0096] The present invention is characterized in that the amount of the
cancer marker of the present invention expressed is corrected by the
correction marker of the present invention. Thus, methods for measuring
the amount of the cancer marker of the present invention expressed and
the amount of the correction marker of the present invention expressed
are by no means limited. For the correction marker of the present
invention, for example, the method may be any method that can detect each
miRNA as described above, as in the case of the cancer marker of the
present invention.

[0097] <Correction Reagent>

[0098] As described above, the correction reagent of the present invention
includes a miRNA detection reagent for detecting hsa-miR-638 and is
characterized in that it is used to correct the expression level of the
cancer marker of the present invention in a biological sample in the
evaluation method of the present invention. According to such a
correction reagent, the correction method of the present invention can be
carried out conveniently.

[0099] The present invention is characterized in that, as described above,
hsa-miR-638 is detected as a correction marker, and a method for
detecting the miRNA is by no means limited. Therefore, it is only
necessary that the miRNA detection reagent contained in the correction
reagent of the present invention can detect this miRNA as described
above, and for example, the kind, composition, etc. of the reagent are by
no means limited. Furthermore, those skilled in the art can set a miRNA
detection reagent for detecting this miRNA as appropriate based on common
general technical knowledge.

[0100] The correction reagent of the present invention may contain any of
various enzymes, buffer solutions, washing solutions, dissolving
solutions, dispersions, diluents, and the like, for example, depending on
various detection methods for detecting the miRNA. Furthermore, the form
of the correction reagent of the present invention is not particularly
limited. For example, it may be a wet-type reagent in the liquid form or
a dry-type reagent in the dry form.

[0101] The miRNA detection reagent is not particularly limited, and
examples thereof include reagents to be used in the Northern blot
analysis and real-time RT-PCR detection method described above. Specific
examples of the miRNA detection reagent to be used in the Northern blot
analysis include labeled probes that can hybridize to either of these
miRNAs, such as those described above. Furthermore, specific examples of
the reagent to be used in the real-time RT-PCR detection method include
primers for amplifying cDNA from total RNA, primers for amplifying either
of these miRNAs, fluorescent reagents that specifically bind to or
intercalate into double-stranded nucleic acids, and various reagents that
can be used in nucleic acid amplification, such as those described above.
Examples of the various reagents include nucleotide triphosphate (dNTP)
and enzymes such as DNA polymerase.

EXAMPLES

[0102] Next, the present invention will be described with reference to
examples. It is to be noted, however, the present invention is by no
means limited by the following examples.

Example 1

[0103] Bloods were collected from a normal subject and cancer patients
shown in the following tables, and a fraction containing serum and plasma
(hereinafter referred to as a sample) was separated from each blood. From
the thus-obtained sample, total RNA was extracted using Isogen-LS (trade
name) (NIPPON GENE CO., LTD.), and the concentration of the total RNA was
adjusted so as to be 100 ng/μl. Then, the total RNA was
dephosphorylated using alkaline phosphatase derived from calf small
intestine (trade name "Alkaline Phosphatase (Calf intestine) (CIAP)",
TAKARA BIO INC.). Thereafter, the total RNA was labeled with a cyanine
dye using ligase (trade name "T4 RNA Ligase (Cloned)", Ambion). Note here
that these operations were carried out using a kit (trade name "miRNA
Labeling Reagent and Hybridization Kit", catalog No. 5190-0408, Agilent
Technologies, Inc.) in accordance with a protocol attached thereto.
Furthermore, using a microarray slide (trade name "Human miRNA Microarray
kit 8×15K V2", catalog No. G4470B, Agilent Technologies, Inc.),
hybridization of the cyanine labeled total RNA was caused, and signals
were scanned using a scanner (trade name "DNA Microarray Scanner",
Agilent Technologies, Inc.). For the signal detection, software programs
"Feature Extraction 9.5.3 Software and Agilent Scan Control Software
(ver.7.0)" accompanying the scanner were used.

[0104] In the above-described manner, signal values indicating the
expression levels of hsa-miR-92a and hsa-miR-494 as the cancer markers
and hsa-miR-638 as the correction marker in the sample of each of the
subjects were obtained. Still further, with regard to each subject, the
following (A) and (B) were calculated.

(A) the ratio between the expression level of the cancer marker and the
expression level of the correction marker, represented by the following
formulae:

[0105] hsa-miR-92a/hsa-miR-638 (hereinafter, "92a/638")

[0106] hsa-miR-494/hsa-miR-638 (hereinafter, "494/638")

(B) a relative value obtained by dividing the ratios "92a/638" and
"494/638" of each of the subjects respectively by ratios "92a/638" and
"494/638" of the normal subject (BS63), represented by the following
formulae:

[0109] The results thereof are shown in Table 1 and Table 2 below,
respectively. Table 1 shows the result obtained when the cancer marker
was hsa-miR-92a and the correction marker was hsa-miR-638. Table 2 shows
the result obtained when the cancer marker was hsa-miR-494 and the
correction marker was hsa-miR-638.

[0110] As can be seen from Table 1, it was found that, when hsa-miR-92a
was detected as the cancer marker and corrected with hsa-miR-638 as the
correction marker, the expression levels 92a/638/BS63 in the cancer
patients were from 0.022 to 0.665, which were much lower than "1.0" as
the expression level 92a/638/BS63 in the normal subject. Also, as can be
seen from Table 2, it was found that, when hsa-miR-494 was detected as
the cancer marker and corrected with hsa-miR-638 as the correction
marker, the expression levels 494/638/BS63 in the cancer patients were
from 0.03 to 0.086, which were much lower than "1.0" as the expression
level 494/638/BS63 in the normal subject. These results demonstrate that,
by detecting the cancer marker of the present invention, the presence or
absence of canceration can be judged with high reliability.

Example 2

[0111] Bloods were collected from acute myelogenous leukemia (AML)
patients (n=2) and normal subjects (n=7), and a fraction containing serum
and plasma (hereinafter referred to as a "sample") was recovered by
centrifuging each of the bloods at 15,680 m/s2 for 15 minutes.
Except that the thus-obtained samples were used, the expression of miRNAs
in each sample was analyzed by microarray analysis in the same manner as
in Example 1. Then, with regard to each of the samples derived from the
normal subjects, the signal value ranks (Individual rank_Normal) of the
various miRNAs expressed were determined, and also the signal value
average ranks (Average rank_Normal) of the various miRNAs among the
samples derived from the normal subjects were determined. Furthermore, in
order to examine the variation in expression of each miRNA among the
samples, the signal value rank of each of the various miRNAs in each of
the samples derived from the normal subjects was divided by the signal
value average rank of the corresponding miRNA among the samples derived
from the normal subjects, and the logarithm thereof
(log10(individual rank_Normal/Average rank_Normal]) was determined
(The base was 10).

[0112] Using these results, first, with regard to the samples prepared
from seven specimens derived from the normal subjects, expression
profiles of various miRNAs were compared with each other. The result
thereof is shown in FIGS. 1A and 1B. FIG. 1A shows the signal intensity
values of the various miRNAs contained in each of the samples prepared
from the seven specimens. In FIG. 1A, 1 to 7 on the horizontal axis
indicate the respective specimens, and the vertical axis indicates the
signal intensity. In FIG. 1A, "hsa-miR" is represented by "miR"
(hereinafter the same). FIG. 1B shows a logarithmic value
(log10[individual rank_Normal/Average rank_Normal]) of a value
obtained by dividing the rank of each of the various miRNAs in the seven
normal subjects by the average rank thereof. In FIG. 1B, the horizontal
axis indicates the respective miRNAs, and the vertical axis indicates the
logarithmic value.

[0113] As a result, as can be seen from FIG. 1A, in any of the samples
prepared from the seven specimens derived from the normal subjects,
expression of hsa-miR-638 and hsa-miR-92a was observed, and further, it
was found that hsa-miR-638 exhibited the highest expression level.
Furthermore, as can be seen from FIG. 1B, with regard to hsa-miR-638, the
above-described logarithmic value regarding the signal value rank was 0.
The absolute value of this logarithmic value varies greatly about 0 as
the difference in behavior of the expression profile of the miRNA among
the respective samples of the normal subjects becomes noticeable, for
example. However, since this logarithmic value was "0" with regard to
hsa-miR-638, it was found that hsa-miR-638 exhibits similar expression
profile in any of the samples of the normal subjects. These results
demonstrate that hsa-miR-638 serves as a correction marker in
quantification of miRNA in a sample.

[0114] Next, with regard to the samples derived from the AML patients, the
signal value average ranks (Average rank_AML) of the various miRNAs
between the samples were determined. Then, the signal value average rank
(Average rank_AML) of each of the various miRNAs between the samples
derived from the two AML patients was divided by the signal value average
rank (Average rank_Normal) of each of the various miRNAs among the
samples derived from the seven normal subjects to compare the expression
profiles of the various miRNAs in the samples derived from the AML
patients. The result thereof is shown in FIG. 2. FIG. 2 is a graph
showing the ratio (Average rank_AML/Average rank_Normal) between the
signal value average rank of each miRNA between the samples derived from
the AML patients and the signal value average rank of each miRNA among
the samples derived from the normal subjects. As the value of this ratio
becomes greater, it means that the difference in expression profile is
noticeable between the patients and the normal subjects. In FIG. 2, the
vertical axis indicates Average rank_AML/Average rank_Normal, and the
horizontal axis indicates the respective miRNAs.

[0115] As can be seen from FIG. 2, the signal value average rank of
hsa-miR-92a in the AML patients was about 20 times greater than that of
hsa-miR-92a in the normal subjects. This indicates that the expression of
hsa-miR-92a decreased markedly in the AML patients. This result
demonstrates that hsa-miR-92a can serve as a cancer marker for AML.

Example 3

[0116] From each of 77 subjects consisting of 39 men and 38 women, a
fraction containing plasma and serum (hereinafter referred to as a
"sample") was recovered in the same manner as in Example 2. Among these
subjects, 16 subjects were normal subjects and 61 subjects were acute
leukemia patients. The FAB classification of the acute leukemia patients
is as follows:

[0117] The expression levels of hsa-miR-92a and hsa-miR-638 in each of the
samples were analyzed by quantitative RT-PCR using a commercially
available TaqMan (trademark) MicroRNA Assay (Applied Biosystems). Unless
otherwise stated, the analysis was conducted in accordance with the
instructions for use accompanying the commercially available reagent
used.

[0118] First, the total RNA was isolated from the sample derived from each
of the subjects in the same manner as in Example 2. With 20 ng of the
total RNA as a template, a reverse transcription reaction was carried out
using "TaqMan (trademark) MicroRNA RT Kit" (trade name) (Applied
Biosystems). Specifically, 20 ng of the total RNA (input RNA) were added
to 15 μl of a reverse transcription reaction solution having the
following composition, and the resultant mixture was incubated at
16° C. for 30 minutes, at 42° C. for 30 minutes, and at
85° C. for 5 minutes, thereby causing a reverse transcription
reaction. Note here that, in the following reaction solution, the
following primer for hsa-miR-92a and primer for hsa-miR-638 were both
miRNA-specific stem-loop primers (Looped RT primers). Specifically, as
the following primer for hsa-miR-92a, Assay Name hsa-miR-92, Product
number 4373013 of TaqMan (trademark) MicroRNA Assay (Applied Biosystems)
was used, and as the primer for hsa-miR-638, Assay Name hsa-miR-638,
Product number 4380986 of TaqMan (trademark) MicroRNA Assay (Applied
Biosystems) was used.

[0119] Subsequently, with a transcription product obtained through the
reverse transcription reaction as a template, quantitative real-time PCR
was carried out using Mx3005P (trademark) QPCR system (STRATAGENE). As
the reverse transcription product, 1 μl of diluted solution of the
reverse transcription product obtained by mixing the reaction solution
after the above-described reverse transcription reaction and
nuclease-free water at a ratio of 1:2 (volume ratio) was used.
Specifically, 20 μl of a PCR reaction solution having the following
composition to which 1 μl of the diluted solution had been added was
treated at 95° C. for 2 minutes and then was subjected to 50
cycles of incubation with a treatment at 95° C. for 15 seconds and
60° C. for 1 minute as one cycle. In the following PCR reaction
solution, the following buffer contained a primer set included in the
above-described TaqMan (trademark) MicroRNA Assay (Assay Name hsa-miR-92:
Product number 4373013: Applied Biosystems) for hsa-miR-92a detection and
TaqMan (trademark) Probe. Alternatively, the buffer contained a primer
set included in the above-described TaqMan (trademark) MicroRNA Assay
(Assay Name hsa-miR-638: Product number 4380986: Applied Biosystems) for
hsa-miR-638 detection and TaqMan (trademark) Probe. In the probe for
detecting hsa-miR-92a and the probe for detecting hsa-miR-638, the 5' end
was labeled with FAM and the 3' end was labeled with TAMRA.

[0120] Data obtained through the quantitative RT-PCR was analyzed with
MxPro-Mx3005P (trademark) version 3.00 (STRATAGENE), and the automatic Ct
setting for baseline adaptation and threshold for Ct determination also
were conducted. Then, with regard to the sample derived from each
subject, the ratio (hsa-miR-92a/hsa-miR-638) between the fluorescence
intensity indicating hsa-miR-92a as the cancer marker and the
fluorescence intensity indicating hsa-miR-638 as the correction marker
was determined. Furthermore, the Mann-Whitney U test was conducted to
examine the statistical significance of the difference between the ratios
determined from the samples derived from the normal subjects and the
ratios determined from the samples derived from the acute leukemia
patients.

[0121] The result thereof is shown in FIG. 3. FIG. 3 is a graph showing
the fluorescence intensity ratio (hsa-miR-92a/hsa-miR-638) with regard to
the samples derived from the normal subjects and the samples derived from
the acute leukemia patients. As can be seen from FIG. 3, with regard to
the samples derived from the normal subjects, the fluorescence intensity
ratios were as follows: median=0.4672; 25%=0.0051; 75%=0.0138; maximum
value=2.3388; minimum value=0.0189; and average value=0.74587. On the
other hand, with regard to the samples derived from the acute leukemia
patients, the fluorescence intensity ratios were as follows:
median=0.0129; 25%=0.0058; 75%=0.0274; maximum value=0.1085; minimum
value=0.0004; and average value=0.020585. From this result, it was found
that hsa-miR-92a expression decreased markedly in the samples derived
from the patients. Furthermore, P value, which indicates the statistical
significance of the difference between the normal subjects and the acute
leukemia patients, was 2.0×10-8, from which it was found that
the difference was significant enough. The result described above
demonstrates that hsa-miR-92a in serum or plasma is a particularly highly
sensitive marker for acute leukemia.

Example 4

[0122] With regard to two pancreas cancer patients, hsa-miR-92a and
hsa-miR-638 were measured in the same manner as in Example 3 before and
after operation, and the fluorescence intensity ratio
(hsa-miR-92a/hsa-miR-638) was calculated. As a result, relative values of
the fluorescence intensity ratio after operation in the two patients
assuming that the fluorescence intensity ratio before operation was 1
were 2.67 and 1.40, respectively. It can be seen from this result that
the amount of hsa-miR-92a expressed decreases when a subject has pancreas
cancer, and the amount of hsa-miR-92a expressed increases after the
pancreas cancer is treated. That is, it can be said that it is possible
to judge whether or not a subject has pancreas cancer based on the amount
of hsa-miR-92a expressed.

[0123] Similarly, also with regard to four liver cancer patients,
hsa-miR-92a and hsa-miR-638 were measured in the same manner as in
Example 3 before and after operation, and the fluorescence intensity
ratio (hsa-miR-92a/hsa-miR-638) was calculated. As a result, relative
values of the fluorescence intensity ratio after operation in the four
patients assuming that the fluorescence intensity ratio before operation
was 1 were 6.13, 12.6, 156.52, and 12.12, respectively. It can be seen
from this result that the amount of hsa-miR-92a expressed decreases when
a subject has liver cancer, and the amount of hsa-miR-92a expressed
increases after the liver cancer is treated. That is, it can be said that
it is possible to judge whether or not a subject has liver cancer based
on the amount of hsa-miR-92a expressed. It is to be noted that, with
regard to other cancers, the similar results were obtained.

INDUSTRIAL APPLICABILITY

[0124] According to the present invention, by detecting the expression
level of the cancer marker of the present invention in a biological
sample, it becomes possible to judge the presence or absence of the
development or progression of cancers with high reliability, for example.
Furthermore, the cancer marker of the present invention provides a marked
difference between negative and positive regarding the canceration, for
example. Thus, according to the cancer marker of the present invention,
it is possible to detect cancers at an initial stage whereas such
detection is difficult by general palpation and the like